Any one have experienced to conjugate peptide with dye? I have protected peptide and i want to conjugate with dye which has NHS ester. I am confused because for conjugating the condition should be base but for deprotection should be acid. If I conjugate first I worry when i deprotect protection group in acid condition it will be cleave dye. But if I deprotect protection group my dye will not conjugate in potition that i want since there are 2 amine primer in my peptide. Any the best way to resolve this problem?
Thank you
The most elegant way to label the peptide is performed during the synthesis, e.g. by attaching the dye n-terminally. However, many dyes are stable against acidic deprotection reagents. "Reactive fluorescein, sulforhodamine B, tetramethylrhodamine, coumarin, eosin, dabcyl, dabsyl, or biotin labels, as well as several of our new atto labels, should be stable enough to resist the harsh deprotection conditions." (see link) https://www.sigmaaldrich.com/technical-documents/articles/chemfiles/fluorescent-labeling.html
Conjugate peptide with dye one must know which dye is to be connected . The role of the functional important during attachment where one has to attach through azo , -OH, Amino , COOH group. Protection of the functional group technique will be useful in this case.Structure of peptide is also important for attachment of dye either NH or CO end.
Your procedure will strongly depend on the dye you intend to use and also on the peptide. The easiest that can be done is to use a carbodiimide. This techniques allows you to produce covalent bonds between amino- and carboxyl-groups.
Good luck.
I have no experience with this label. You should ask the manufacturer of the reagent for advice. BTW: The protonation of the dye is not a problem, because this is reversible. However, your deprotection conditions may lead to degradation of the dye.
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